The overall goal of this project is to test the hypothesis that reduced Cx43 expression and subsequent altered cell-cell communication triggered by high glucose/diabetes leads to cell death and breakdown of vascular homeostasis in the retinal capillaries in diabetic retinopathy. The hypothesis is based on findings that high glucose reduces Cx43 expression in microvascular endothelial cells and retinal pericytes and compromises gap junction intercellular communication. Our current study showed reduced Cx43 expression triggers apoptosis (manuscript under revision, IOVS). The overall working hypothesis is that reduced Cx43 expression triggers vascular cell death, a prominent and early lesion associated with the development of diabetic retinopathy, which in turn, reduces cell-cell communication in the retinal vascular cells and ultimately disrupts vascular homeostasis. Retinal vascular cell death is known to occur by apoptosis but it is unknown how apoptosis is triggered during the development of diabetic retinopathy. Preliminary data suggests that communication between vascular cells, that is endothelial cell-endothelial cell, endothelial cell-pericyte, and pericyte-pericyte is essential for their survival, and that disruption in cell-cell communication may trigger apoptosis and interfere with their role to form a functional unit via the connexin (Cx) channels in cell junctions. The specific focus of this proposal is to determine (i) the effect of reduced Cx43 expression in retinal endothelial cells and pericytes, (ii) whether high glucose-induced excess ECM synthesis modulates Cx43 expression in retinal endothelial cells and pericytes, and (iii) whether reduced Cx43 expression induces tight junction dysfunction in the retinal endothelial cells. A variety of cell biological, molecular biological and biochemical techniques including antisense oligo mediated specific downregulation of gene expression, scrape load dye transfer technique, fluorescence microscopy and Western blot method will be used for studying the consequence of high glucose-induced inhibition of Cx43 expression and reduced cell-cell communication on retinal cell viability and function. Findings from these studies will provide a better understanding of cell-cell communication underlying altered Cx43 expression and gap junction channels in retinal vascular cells and their role in breakdown of vascular homeostasis associated with diabetic retinopathy. An important mission of the agency is to find a cure and prevent complications arising from diabetic retinopathy. The proposed project is expected to provide important findings that can help in better understanding the pathogenesis of diabetic retinopathy. PUBLIC HEALTH RELEVANCE: Currently, there is no cure for diabetic retinopathy, the leading cause of blindness in the working age Americans. It is expected that the findings from this project would provide valuable insight towards testing a novel mechanism underlying retinal vascular cell loss and capillary leakage, the two critical steps in the development of diabetic retinopathy.